Transformed centrifugation at 10,000×g and resuspended in

Transformed bacteria with CPG2 and TAT-CPG2 constructs were
grown in the LB medium containing 100 µg/ml ampicillin at 37 ?C to reach an optical density of
0.6 at 600 nm. ExpressionM1  of the fusion proteins was induced at 0.5 mM and 1 mM of IPTG. Cells
were grown at either 37 ?C or 28 ?C for 4 h. To increase the level of soluble TAT-CPG2,
the induction temperature and IPTG concentration for protein
expression were optimized. Experiments were carried out using
0.5 and 1 mM   IPTG
and the induction
process was performed at 28 ?C and 37 ?C. Purification
of CPG2 and TAT-CPG2 fusion proteins were carried out under native and denaturing
conditions by the batch method of Qiagen. For purification of recombinant
proteins under native condition the bacterial cells
were harvested by centrifugation at 10,000×g and resuspended in binding buffer
(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole,
and 1 mM PMSF at pH 8). Lysozyme was added at a concentration of 0.1 mg/ml and
incubated on ice for 30 min. Cells were then disrupted by sonication (6 ×10 s
bursts at 200–300 W with a 10 s cooling period in-between)
on ice. The bacterial lysates were centrifuged (10,000×g at 4 °C for 30 min), and
the supernatants were added to a 50% Ni-NTA resin pre-equilibrated with binding
buffer and mixed gently by shaking (200 rpm on a rotary shaker) at 4 °C for 60
min. The lysate–Ni-NTA mixture was loaded on an empty
PD10 column. The column was washed twice with 4 ml wash buffer (50 mM NaH2PO4,
300 mM NaCl, 20 mM imidazole, pH 8). The CPG2 and TAT-CPG2 fusion proteins were
eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl,
250 mM imidazole, pH 8).  Eluted proteins
were desalted on a PD10 desalting column. For purification under denaturing condition, pellets
from purification under native condition were resuspended in buffer B (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M
urea, pH 8) and stirred for 60 min at room temperature. The mixture was then
centrifuged at 10,000×g for 30 min at room temperature to pellet the cellular
debris. Supernatant was mixed with 50% Ni-NTA resin and gently shaked (200 rpm
on a rotary shaker) at room temperature for 60 min. The lysate–Ni-NTA mixture was
added to a column and washed twice with 4 ml buffer C (100 mM NaH2PO4,
10 mM Tris-HCl, 8 M urea, pH 6.3). The recombinant proteins were eluted 4 times
with 0.5 ml buffer D (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M
urea, pH 5.9), followed by 4 times elution with 0.5 ml buffer E (100 mM NaH2PO4,
10 mM Tris-H, 8 M urea, pH 4.5). Monomers generally elute in buffer D, while
multimers and aggregates elute in buffer E. The eluted proteins were desalted
by PD10 desalting column. The purified fusion proteins were verified by
SDS/PAGE, coomassie brilliant blue staining and western blot analysis with
anti-His6-peroxidase antibody (1:500; Roche). The protein concentrations were
estimated by the Bradford method (Bradford, 1976).

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